Antibiotic resistant and pathogenic Gram-negative bacteria are an increasingly important public health concern and are expected to soon surpass methicillin-resistant S. aureus as the principal cause of mortality due to bacterial infection. Despite evident need, new antibiotics are not being developed at an adequate rate and most effort involves modifications of existing drugs, rather than identification and development of novel drug targets. An attractive target for the much-needed development of new antibiotic therapeutics is the Type Three Secretion System (T3SS), a virulence factor delivery machine that is conserved among over 25 species of Gram-negative bacteria (including category A, B, and C pathogens). The T3SS is a multi-protein needle-like machine that spans the bacterial and host membranes and delivers protein translocator molecules into the membrane of the target cell and effector molecules into the cytosol of the target cell. The effectors promote virulence by co-opting cellular processes and subverting host defenses. While the molecular mechanisms of TTSS regulation are largely unknown, key requirements are that the pore is constitutively closed, that it opens in response to a stimulus, and secretion is an orderly, hierarchical process. Effectors are secreted directly into the host cell, through a preformed translocon pore. This pore is composed of secreted translocator proteins that must be secreted prior to effectors. A key regulatory protein is the plug which blocks the pore. Following plug protein secretion translocators are secreted, followed by effectors. In strains where plug proteins have been deleted, effectors are secreted constitutively, translocator secretion is severely defective, and the strains are non-virulent. The origin of the essential translocator-effector hierarchy is unknown. We have recently determined the first structure of a plug protein bound to a chaperone for a translocator. This structure reveals that plugs are molecular scaffolds that are tethered to translocators. We intend to further elucidate the role of plug-translocator scaffolding in multiple gram-negative species, and to understand the novel effector function of the Chlamydial plug protein. We have shown these proteins to possess novel tubulin binding function are poised, with our recent structure, to determine the molecular strategies that Chlamydia use to regulate the host's microtubule cytoskeleton. Finally, we will evaluate the chaperone-translocator interaction as a novel therapeutic target for the development of broad-spectrum antibiotics.